Agents currently used to treat HIV infections attempt to block replication of the HIV virus by blocking the reverse transcriptase or by blocking the HIV protease. Three categories of anti-retroviral agents in clinical use are nucleoside analogs (such as AZT), protease inhibitors (such as nelfinavir), and the recently introduced non-nucleoside reverse transcriptase inhibitors (NNI) such as nevirapine.
The recent development of potent combination anti-retroviral regimens have significantly improved the prognosis for persons with HIV and AIDS. Combination therapies may be a significant factor in the dramatic decrease in deaths from AIDS (death rate as well as absolute number). The most commonly used combinations include two nucleoside analogs with or without a protease inhibitor.
Nevirapine is currently the only NNI compound which has been used in combination with AZT and/or protease inhibitors for the treatment of HIV. A new series of effective drug cocktails will most likely involve other NNIs in combination with nucleoside and protease inhibitors as a triple action treatment to combat the growing problem of drug resistance encountered in single drug treatment strategies.
The high replication rate of the virus unfortunately leads to genetic variants (mutants), especially when selective pressure is introduced in the form of drug treatment. These mutants are resistant to the anti-viral agents previously administerd to the patient. Switching agents or using combination therapies may decrease or delay resistance, but because viral replication is not completely suppressed in single drug treatment or even with a two drugs combination, drug-resistant viral strains ultimately emerge. Triple drug combinations employing one (or two) nucleoside analogs and two (or one) NNI targeting RT provide a very promising therapy to overcome the drug resistance problem. RT mutant strains resistant to such a triple action drug combination would most likely not be able to function.
Dozens of mutant strains have been characterized as resistant to NNI compounds, including L1001, K103N, V106A, E138K, Y181C and Y188H. In particular, the Y181C and K103N mutants may be the most difficult to treat, because they are resistant to most of NNI compounds that have been examined.
Recently, a proposed strategy using a knock-out concentration of NNI demonstrated very promising results. The key idea in this strategy is to administer a high concentration of NNI in the very beginning stages of treatment to reduce the virus to undetectable levels in order to prevent the emergence of drug-resistant strains. The ideal NNI compound for optimal use in this strategy and in a triple action combination must meet three criteria:
1) very low cytotoxicity so it can be applied in high doses;
2) very high potency so it can completely shut down viral replication machinery before the virus has time to develop resistant mutant strains; and
3) robust anti-viral activity against current clinically observed drug resistant mutant strains.
Novel NNI designs able to reduce RT inhibition to subnanomolar concentrations with improved robustness against the most commonly observed mutants and preferably able to inhibit the most troublesome mutants are urgently needed. New antiviral drugs have the following desired characteristics: (1) potent inhibition of RT; (2) minimum cytotoxicity; and (3) improved ability to inhibit known drug resistant strains of HIV. Currently, few anti-HIV agents possess all of these desired properties.
Two non-nucleoside inhibitors (NNI) of HIV RT that have been approved by the US Food and Drug Administration for licensing and sale in the United States are nevirapine (dipyridodiazepinone derivative) and delavirdine (bis(heteroaryl)piperazine (BHAP) derivative, BHAP U-90152). Other promising new non-nucleoside inhibitors (NNIs) that have been developed to inhibit HIV RT include dihydroalkoxybenzyloxopyrimidine (DABO) derivatives, 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) derivatives, tetrahydrobenzondiazepine (TIBO), 2′,5′-Bis-O-(tert-butyldimethylsilyl)-3′-spiro-5″-(4″-amino-1″,2″-oxathiole-2″,2′-dioxide)pyrimidine (TSAO), oxathiin carboxanilide derivatives, quinoxaline derivatives, thiadiazole derivatives, and phenethylthiazolylthiourea (PETT) derivatives.
NNIs have been found to bind to a specific allosteric site of HIV-RT near the polymerase site and interfere with reverse transcription by altering either the conformation or mobility of RT, thereby leading to a noncompetitive inhibition of the enzyme.
A number of crystal structures of RT complexed with NNIs have been reported (including α-APA, TIBO, Nevirapine, and HEPT derivatives), and such structural information provides the basis for further derivatization of NNI aimed at maximizing binding affinity to RT. However, the number of available crystal structures of RT NNI complexes is limited.
Given the lack of structural information, alternate design procedures must be relied upon for preparing active inhibitors. One such method which provides important information about predicting inhibitor interactions is receptor-targeted molecular modeling which heavily relies on the integrated information from crystal structures. The inclusion of such modeling information in the drug design process is likely to contribute to a more efficient identification of promising non-nucleoside inhibitors of HIV RT.
In the design of novel inhibitors, it is our working hypothesis that by examining multiple crystal structures of RT-NNI complexes can one understand precisely how the NNI pocket can adjust to accomodate the binding of a particular NNI. Our composite binding pocket, unlike a single crystal structure, is able to summarize the nature and extent of the flexibility of the active site residues in the NNI binding site of RT. This allowed the de novo design of PETT compounds after positioning the compounds into the NNI active site of RT.
As described in copending U.S. patent application Ser. No. 09/040,538, two major features observed from the composite binding pocket model are previously unidentified spacious regions and polar regions at the Wing 2 portion of the binding pocket. It was postulated that the spacious or flexible regions of the binding pocket can accommodate and interact favorably with functional groups larger than a pyridyl ring at the Wing 2 region. Polar regions of the binding pocket would interact favorably with properly positioned polar groups on the inhibitor molecule, such as halogen groups.
Using the composite binding pocket model, a series of potent NNI compounds was synthesized and assayed for anti-viral activity. These compounds abrogated HIV replication in HTLVM-infected peripheral blood mononuclear cells at nanomolar concentrations (IC50[p24]=<1 nM) without evidence of cytotoxicity (IC50[MTA]>100 μM. Surprisingly, several compounds also demonstrated high potency against multiple drug resistant mutant strains, as discussed below and claimed herein.